This invention involves a process for removal of pyrogenic material from bovine thrombin.
Pyrogens can cause undesirable reactions in humans, including fever, chills, pains in back and legs and malaise. Because of these undesirable physiological reactions caused by the presence of pyrogens in animals, pharmaceutical preparations must be relatively free of pyrogens.
All biological products for parenteral prevention, diagnosis, or treatment of disease in man must meet specified tests, including a test for the presence of pyrogens. The pyrogen test as described by United States Pharmacopeia, is a biological test in which a fever response in rabbits is used as the criterion. Because the use of thrombin containing pyrogens in a topical application to produce localized clotting can introduce pyrogens into the body fluid systems, or the blood circulating system, such thrombin preparations must meet the same standards involving pyrogens as parenteral pharmaceutical preparations.
Remington's Pharmaceutical Sciences, Fourteenth Edition, pp. 1524-1525 (1970) emphasizes the difficulty of removing pyrogens from pharmaceutical preparations. The article indicates that the preferred procedure is to prevent the introduction of pyrogens into parenteral preparations, rather than attempt to remove them, a task which it indicates . . . "may well be virtually impossible."
J. Pharm. Pharmac., 30: 198 (1978) indicates that methods previously suggested for removing pyrogens from a pharmaceutical dosage form include recrystallization; careful treatment in the presence in dilute alkali, acid or oxidizing agents; adsorption on charcoal, asbestos or other materials; anion exchange chromatography or silicic acid thin layer chromatography. The authors suggest that the above-named methods had value for laboratory usage in a small-scale, but did not provide optimal removal of pyrogens from an unstable drug on a multi-liter scale. The authors removed the pyrogen-contaminants by molecular filtration, which is an expensive procedure.
The prior art is replete with methods of purification of blood factors, including Factor II (prothrombin), Factor IIa (thrombin), and Factor X. The two major methods are ion exchange chromatography and affinity chromatography.
For example, J. Biol. Chem., 243: 112-117 (1968) describes purification of thrombin by a stepwise chromatographic technique on diethylaminoethyl (DEAE) cellulose, to remove plasminogen and plasmin. FEBS Letters, 51(1): 191-194 (1975) describes purification of bovine trypsin and thrombin by affinity chromatography.
The most relevant prior art relating to purification of blood factors appears to be Biochemica. et Biophysica. Acta., 222: 691-695 (1970) and 434: 199-208 (1976).
The 1970 article describes a method of purifying human blood clotting Factor X. Because prothrombin (Factor II) can be converted to thrombin (Factor IIa) by Factor Xa catalysis, the authors were concerned with obtaining Factor X free of other blood clotting factors, including thrombin. The method involved complexing a blue dye-polysaccharide complex and clotting factors and chromatographing. The method is based on the ionic strength-dependent association of clotting factors with the blue dye portion of the complex and subsequent gel filtrations. At low ionic strength, Factors II and IX were eluted in the void volume, indicating that they were complexed with the blue dye-polysaccharide. Factors VII and X were eluted in the included volume, indicating that they were not complexed with the blue dye-polysaccharide complex. The structure of the blue dye used was identified in J. Chromatography, 69: 201-214 (1972) as having a 4-phenylamino-1-amino-anthraquinone structure.
The 1976 article describes purification of Factors II, VII, IX and X from human plasma by the use of the same anthraguinone blue dye coupled to a polymer of polysaccharide. Elution with acetate buffers separated Factor X from Factors II, VII and IX. The authors indicate that one fraction of the column-bound material showed Factor IIa (thrombin) activity, indicating possible activation of Factor II to thrombin.
Both the 1970 and 1976 article describe the purification of Factor X to remove other clotting factors, e.g., II, VII and IX, as impurities. Neither article discloses or suggests the removal of pyrogens from thrombin.
There is a need for a simple and efficient method of removing pyrogens from blood protein products which can be easily adapted to a multi-liter process.